The focus of this study is to investigate mechanisms by which hepatic cells regulate the expression of the LDL receptor (LDLR), particularly at post-transcriptional stages of gene expression. Liver cells are uniquely tailored for our project for the reason that enhanced expression of the LDL receptor can lead to increased levels of cholesterol internalization and excretion through the bile salt pathway. LDL receptor expression is a major determinant of the efficiency with which the liver clears lipoproteins from the plasma. This relationship is clearly seen in individuals afflicted by familial hypercholesterolemia. Normally, LDL receptor is transcriptionally repressed in response to a constant supply of LDL from the serum. We are determined to identify explicit mechanisms by which hepatic expression of LDL receptor is regulated in order to promote the increased uptake and excretion of cholesterol. In previous studies, our lab has shown that protein kinase C (PKC) activation induces LDL receptor mRNA stabilization which is dependent on cytoskeletal association of LDL receptor mRNA. Specifically we are interested in cytoskeletal tethering of LDL receptor mRNA because of its apparent influence on LDL receptor mRNA stability. For Aim I we intend to identify mechanisms contributing to cytoskeletal tethering of the LDL receptor mRNA. Different truncations of the 2727-3157 region will be expressed in stable HepG2 cell lines using (3-globin fusion constructs to control for cytoskeletal localization of mRNAs. Secondly, after the cytoskeletal binding domain is identified we will try to identify proteins involved in cytoskeletal localization of LDL receptor mRNA. Once the protein necessary for cytoskeletal localization of LDL receptor mRNA is identified we propose to validate the function of this protein through siRNA knockdown. For Aim II we intend to characterize the signaling pathwaysthat influence LDL receptor mRNA stabilization. Currently we hypothesize that LDL receptor mRNA stabilization is influenced by PKC activation and MAP kinase mediators. The methods we propose to use for testing this hypothesis include pharmacological inhibitor treatments for PKC coupled with Actinomycin D time course assays to quantify LDLR mRNA half life using real time PCR. Currently we are using results from a Kinexus phosphoprotein screening to identify MAP kianse candidates for inhibitor treatments. Our objective will be to identify the link between PKC activation and MAP kinase activation leading to LDL receptor mRNA stabilization and identification of the map kinase responsive element in the LDL receptor mRNA. These aims will lead to identification of prospective targets for drug therapies that would serve in enhancing cholesterol uptake and excretion through increased expression of LDL receptor mRNA.